Tooth development is a complex schedule of cell division, differentiation, morphogenesis and tissue-specific extracellular matrix biomineralization. The major objective of this research project is to determine how endogenous regulatory factors act as autocrine and/or paracrine mediators to control tissue-specific enamel biomineralization. Based upon preliminary evidence demonstrating that insulin and insulin- like growth factor (IGF-II) significantly increase enamel biomineralization, we propose to test the hypothesis that insulin and/or IGF ligands and their cognate receptors through signal transduction processes control the rates and amount of enamel biomineralization. A simple in vitro organ culture system devoid of exogeneous serum or plasma supplementation provides a controlled model to test our hypothesis. The following three major Specific Aims are designed to test our hypothesis: (i) to identify the timing of expression and tissue localization for insulin, insulin-like growth factors (IGFI-I and II) and their cognate receptors (e.g., insulin receptor, IGF-I and IGF-II receptors, and IGF binding proteins 1 and 2) from cap through crown stages of tooth development; (ii) to determine the level(s) of control for insulin and IGF induction of enamel biomineralization (e.g. transcription, post- transcriptional, translation, post-translational); and (iii) to test the hypothesis that under-expression of insulin and/or IGF ligands and/or their cognate receptors retards enamel biomineralization. Methods employed include microdissection of embryonic mouse molar tooth organs, organ culture using serumless and chemically-defined medium, mRNA phenotyping using RT-PCR, quantitative PCR, antisense translation arrest assays, in situ hybridization, immunocytochemistry, light and electron microscopy, electron diffraction assays, and computer-assisted three- dimensional reconstructions. The long-term goals of this research program are to understand the regulatory mechanisms controlling tooth morphogenesis and enamel biomineralization, and to apply this knowledge to the design of rational strategies to diagnose and treat dental tissue diseases. These studies use embryonic, fetal and neonatal Swiss Webster strain mouse molar tooth organs.